First identification of Tyrophagus curvipenis (Acari: Acaridae) and pathogen detection in Apis mellifera colonies in the Republic of Korea

Mites of the genus Tyrophagus (Acari: Acaridae) are among the most widely distributed mites. The species in this genus cause damage to stored products and crops, and pose a threat to human health. However, the influence of Tyrophagus spp. in apiculture remains unknown. In 2022, a study focusing on the identification of Tyrophagus species within five apiaries was conducted in Chungcheongnam Province, Republic of Korea. Its specific objective was to investigate the presence of Tyrophagus mites in response to the reported high mortality of honey bee colonies in this area. Morphological identification and phylogenetic analysis using the mitochondrial gene cytochrome-c oxidase subunit 1 (CO1) confirmed for the first time the presence of the mite species Tyrophagus curvipenis in a honey bee colony in the Republic of Korea. Two honey bee pathogens were detected in the mite, a viral pathogen (deformed wing virus, DWV) and a protozoal pathogen (Trypanosoma spp.). The presence of the two honey bee pathogens in the mite suggests that this mite could contribute to the spread of related honey bee diseases. However, the direct influence of the mite T. curvipenis on honey bee health remains unknown and should be further investigated.

www.nature.com/scientificreports/ structures of a greenhouse in Portugal, and they 19 recorded that mites occasionally inhabit the flowers of orchids and may feed on their pollen 19 . Several species of Tyrophagus (such as T. putrescentiae, T. curvipenis, T. tropicus, T. debrivorus, T. similis, T. longgior, T. mixtus, T. perniciosus, T. vanheurni, and T. savasi) have been associated with bees 20 . Tyrophagus putrescentiae (Acari: Acaridae) was detected in dead honey bee samples 21 , in the body of bumblebees 22 , and in honey bee hives in Brazil. This suggests the potential of the mites to harm human health upon consumption of the products from contaminated honey bees 23 . No study reports the presence of the remaining nine species of Tyrophagus in honey bees. Acting as potential carriers of pathogens or vectors for pathogen infections, these mites can affect humans indirectly [24][25][26] ; therefore, the role of T. curvipenis and other species of mites infecting honey bees need further studies and evaluation. Identification of Tyrophagus mites has been traditionally based on morphology 12,14,27 . However, this method is laborious due to the small size of Tyrophagus mites, requires good understanding of morphological traits, and it is time-consuming. The molecular method based on the mitochondrial gene cytochrome-c oxidase subunit 1 (CO1) and the nuclear ribosomal internal transcribed spacer 2 (ITS2) was suggested as an alternative tool for species identification of microscopic mites 28 .
Screening for the factors responsible for the honey bee colony loss was carried out in the dead colonies collected from the regions with high colony collapse reported in winter. Here, mites were detected and collected from dead honey bee colonies in the ROK in 2022. The CO1 and ITS2 sequences were analyzed to identify the mite species. In addition, honey bee pathogens carried by the mite were also identified.
Genetic identification of mite species. The family to which this species belongs can be determined based on the morphological characteristics observed under the microscope. Although morphological traits are highly useful for initial identification, accurately distinguishing the exact species can be challenging. To confirm the identification, species-specific primer pairs targeting the CO1 gene and the ITS2 gene regions of Tyrophagus mites were designed. We successfully optimized the PCR reaction conditions for amplifying the CO1 and ITS2 genes. The sequences were then determined through sequence analysis. Amplification of the CO1 and ITS2 regions from adult mite and egg samples produced bands with expected length of 379 and 500 bp, respectively (Fig. 3a). Sequences of the amplified regions were 100% similar between the adult and egg samples. The search of the obtained CO1 sequences against the GenBank database (blast.ncbi.nlm.nih.gov) revealed 100% sequence identity with T. curvipenis originating from Costa Rica (NCBI accession No.: KY986270) and 86.64% similarity with T. putrescentiae (NCBI accession No.: MH262448). The IST2 sequences of T. curvipenis are not available in the NCBI database, and the search of the sequences isolated from the samples against the GenBank database indicated the highest similarity of 91.78% to T. putrescentiae in China (NCBI accession No.: GQ205623.1). In addition, non-specific band (around 1.7 kb long) was seen in the ITS2 amplification from adult sample (Fig. 3b). The unexpected band was extracted for sequencing. However, the result was unidentified due to the poor sequencing result.
Phylogenetic analysis based on the CO1 gene sequences placed the collected mite sister with T. curvipenis isolate AD2025 collected from a bird nest in Costa Rica and both were in the same clade with T. curvipenis isolated from plants (peach and chayote) in Russia and Costa Rica (Fig. 4).   Table S1). Therefore, to identify the possibility of the honey bee pathogen infection in the mites, we conducted pathogen detection using total nucleic acids extracted from the mites. Two honey bee pathogens (DWV and Trypanosoma) were detected in eggs and adult samples of the mite. Additionally, the two pathogens were present in the honey bee samples from the same colonies where the mites were collected. The identity of the pathogens was confirmed using reverse transcription real-time PCR (RT-qPCR), which produced bands of 199 bp and 276 bp for DWV and Trypanosoma, respectively (Figs. 5 and 6).

Discussion
In this study, Tyrophagus mites were detected and identified in the colonies of five apiaries in Gongju, Chungcheongnam Province, Republic of Korea. This is the first report of T. curvipenis in A. mellifera colonies in the country. Morphological identification and genetic identification using CO1 gene revealed that the mite belongs to the species T. curvipenis, and phylogenetic analysis of the obtained sequence indicated a close relationship between the T. curvipenis from the Republic of Korea and the isolate from a bird's nest in Costa Rica (Fig. 4). In addition, the species phylogeny of Tyrophagus based on nucleotide similarity in the CO1 gene region showed that the T. curvipenis detected in honey bee in this study was in the same cluster as that identified in plants such as peach and chayote from Russia and Costa Rica. The result suggests that the source of T. curvipenis identified in this study could be a flower visited by the honeybee for pollen and nectar collection. Four of the 35 identified species in the genus Tyrophagus have been reported to date in the Republic of Korea: T. putrescentiae, T. similis, T. neiswanderi, and T. longior [29][30][31] . Tyrophagus curvipenis reported here is thus a newly recorded species in the ROK. Similar to other species, T. curvipenis has been found in honey bee hives in New Zealand 32 . It was reported that  www.nature.com/scientificreports/ T. putrescentiae in Korea (Wanju-gun, Jeonnam) inhabits beehives and feeds on debris and pollen 33 . However, the relationship between honey bee health and the presence of T. curvipenis mite in the hive remains unknown. The molecular method was successfully used to identify Tyrophagus species. The result of the molecular method based on the CO1 gene sequence was consistent with the identification based on morphological characteristics. However, due to the limited availability of Tyrophagus sequences in the NCBI database, species identification of the Tyrophagus mite could not be confirmed using the IST2 region. In addition, the IST2 primer pair amplified a non-specific band using the total DNA extracted from adult mites (Fig. 3b). The CO1 gene, owing to a larger deposited sequence database, is potentially more beneficial for species identification of Tyrophagus mites.
Infestation of honey bees with hemolymph-feeding mites such as Varroa and Tropilaelaps increased colony loss over the winter season and transmission of honey bee diseases 4,6,7,[34][35][36] . The viral, bacterial, and fungal honey bee pathogens harbored and transmitted by Varroa and Tropilaelaps mites have been demonstrated 36,37 . The T. curvipenis mite detected in this study was positive for a honey bee viral pathogen (DWV) and protozoal pathogen (Trypanosoma). The infection with these pathogens weakens the colony and increases the winter mortality of honey bees [38][39][40] . Additionally, these pathogens were detected in the honey bee samples where the mites were collected, suggesting a high possibility of mite transmitting such pathogens in the hives. Furthermore, Tyrophagus mites, commonly known as storage mites, feed on mold found in food 41,42 . However, the food sources of T. curvipenis in honey bee hives remain unknown. Therefore, understanding the influence of T. curvipenis mite on honey bees would help to develop appropriate methods for mite prevention and control. In addition, it is necessary to minimize the potential effects of the mites on humans who consume the honey bee products collected from infested colonies, because some Tyrophagus mites were identified as allergens in animals and humans 43,44 . With an observed infestation rate of 100% and the potential to serve as intermediate disease vectors within honey bee colonies in the five apiaries studied, Tyrophagus mites potentially play an important role in the observed colony losses. The findings from this study have the potential to contribute to the development of targeted management strategies aimed at minimizing the impact of Tyrophagus mite infestation on honey bee health and improving overall colony survival rates. By understanding the significance of Tyrophagus mites as potential contributors to colony decline, proactive measures can be implemented to mitigate their negative effects and safeguard the well-being of honey bee populations. This is the first record of T. curvipenis mites in honey bee colonies in the Republic of Korea, and this study demonstrated the presence of honey bee pathogens (DWV and Trypanosoma) in mites. The result suggests that the mite could have an important role in spreading the honey bee pathogens. However, whether T. curvipenis mite contributed to the death of honey bee colonies has not been confirmed in this study; therefore, further study is necessary to understand the influence of this enemy and biological vector of honey bee pathogens in apiculture, and to reveal the potential risk of mites to humans consuming the products collected from infested honey bee colonies.

Materials and methods
Mite collection and morphological identification. Honey bee samples were collected from dead colonies in apiaries in Gongju-si, Chungcheongnam Province, ROK, in November 2022. A total of 45 honey bee samples were collected from 15 colonies in five different apiaries for mite examination. The presence of mites was confirmed under a dissecting microscope. The mites were collected from the body surface and hairs of honey bees. Mites were mounted on slides with Hoyer's medium. The specimens were identified and measured according to Fan and Zhang 12 . All the measurements used herein are in micrometers. After species identification, the mites were used for genetic analysis and pathogen detection.
Genomic DNA extraction and total nucleic acid extraction. Honey bee samples and collected mites were washed three times using UltraPure™ distilled water (Invitrogen, USA) and used for total nucleic acid extraction. The Maxwell RSC viral total nucleic acid purification Kit (Promega, USA) was used for nucleic acid extraction according to the manufacturer's instructions. The extracted nucleic acids were used for the detection of honey bee pathogens. Extraction of the mite genomic DNA (gDNA) was done using a QIAamp DNA Mini Kit (Qiagen, UK) according to the manufacturer's instructions with some modifications 45 . Ten adult mites or four mite eggs collected from each honey bee colony were pooled and placed in a 1.5 mL tube containing 200 µL ATL buffer and 2.381 mm steel beads (Hanam, ROK). After homogenizing at 5000 rpm for 15 s, 20 µL proteinase K (50 µg/mL) was added, and the mixture was incubated at 56 °C for 1 h. Exactly 200 µL of lysis buffer was added to the solution, and the solution was incubated for 10 min at 70 °C. Next, 200 µL of 98% ethanol was added, and the mixture was mixed by vortexing. The solution was transferred to the AIAamp mini spin column and centrifuged at 12,000×g for 1 min. After washing the spin column twice with the washing buffer, the genomic DNA of the mites was eluted into a new tube using the elution buffer and centrifuged at 12,000×g for 2 min. The isolated DNA was used for PCR amplification of the CO1 and ITS2 regions.
Detection of honey bee pathogens. The mite and honey bee samples from each colony were tested for the presence of honey bee pathogens using the total nucleic acid of honey bee and mite samples.  PCR amplification and sequencing of mite DNA. The CO1 and ITS2 regions of mites were amplified using universal primer sets CO1-forward (5ʹ-GTT TTG GGA TAT CTC TCA TAC-3ʹ) and CO1-reverse (5ʹ-GAG CAA CAA CAT AAT AAG TATC-3ʹ) 46 ; and ITS2-forward (5ʹ-CGA CTT TCG AAC GCA TAT TGC-3ʹ) and ITS2reverse (5ʹ-GCT TAA ATT CAG GGG GTA ATC TCG -3ʹ) 47 . Each reaction mixture (20 µL) was composed of 20 ng of gDNA template, 1 µL of each primer (10 pmol), AccuPower® PCR preMix and master Mix (Bioneer, Korea), and ddH 2 O (to make up to 20 µL). The PCR thermal cycler protocol was optimized as follows: 94 °C (5 min Phylogenetic analysis. Identified nucleotide sequences from different adult and egg samples were subjected to BLASTn search against the NCBI nucleotide database. The nucleotide sequences were aligned using BioEdit and Cluster W software 48,49 . The phylogenetic tree based on the CO1 sequence was created using the neighbor-joining method with 1000 bootstrap replications 50 in MEGA7 software 51 .

Data availability
All data generated or analyzed during the current study are available in the National Center for Biotechnology Information (NCBI) repository, accession number: OQ121480 (ITS2) and OQ121363 (CO1).